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Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line

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Item Type:Article
Title:Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line
Creators Name:Chu, V.T. and Graf, R. and Wirtz, T. and Weber, T. and Favret, J. and Li, X. and Petsch, K. and Tran, N.T. and Sieweke, M.H. and Berek, C. and Kühn, R. and Rajewsky, K.
Abstract:Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells.
Keywords:CRISPR/Cas9, sgRNA Design, Knockout Efficiency, Primary Cells, B Cells, Animals, Mice
Source:Proceedings of the National Academy of Sciences of the United States of America
ISSN:0027-8424
Publisher:National Academy of Sciences (U.S.A.)
Volume:113
Number:44
Page Range:12514-12519
Date:1 November 2016
Official Publication:https://doi.org/10.1073/pnas.1613884113
PubMed:View item in PubMed

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